Phospholipid biosynthesis in Saccharomyces cerevisiae is regulated in a coordinated fashion. Inositol 1-phosphate synthase [I1PS], Cytidine diphosphate diacylglycerol synthase [CDP-DGS], phosphatidylserine synthase [PSS], phosphatidylserine decarboxylase [PSD] and the phospholipid methyltransferases (PMTs) which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC), are all subject to regulation by inositol and choline (See FIG. 1). All of these enzymes show various degrees of repression in cells grown in the presence of inositol, and all display maximal repression When cells are grown in the presence of inositol and choline. However, the enzyme ultimately responsible for the production of PI, phosphatidylinositol synthase (PIS), is not regulated in response to phospholipid precursors.
Several of the phospholipid biosynthetic enzyme activities have been shown to be regulated at the level of transcription of structural genes. The steady-state levels of INOl mRNA and CHOI mRNA encoding for I1PS and PSS respectively, are reduced when wild type cells are grown in the presence of inositol. The mRNA levels are further reduced when both inositol and choline are present in the growth medium. A similar response has also been observed with the CHO2 and OP13 genes encoding the PMTs (See FIG. 1).
Several of the coregulated structural genes have also been shown to be controlled by a common set of regulatory genes. Many mutants with defects in the regulation of phospholipid metabolism were originally isolated on the basis of a defect in I1PS regulation. The wild-type products of the regulatory genes IN02 and IN04 are required for expression of IN01. Mutations at either of these two loci result in failure of cells to express I1PS, leading to inositol auxotrophy. The ino2 and ino4 mutants are also unable to derepress the entire set of enzymes that are subject to coordinate control by inositol and choline.
Mutants with lesions at the OPIl locus were originally isolated by Greenberg, M., Goldwasser, P., and Henry, S. (1982). Mol. Gen. Genet. 186, 157-163 and Greenberg, M. L., Reiner, B., and Henry, S. A. (1982). Genetics 100, 19-33 on the basis of an Over Production of Inositol Phenotype (Opi.sup.-). The opil mutants constitutively express derepressed levels of I1PS, as well as many of the other coregulated enzymes. The effect of the opil regulatory mutation is also apparent at the level of mRNA. In opil mutants, INOl transcript is constitutively overexpressed regardless of the growth condition. Thus, the opil gene, SEQ ID NO: 1; is believed to encode a negative regulatory factor that is required to repress the whole subset of enzymes that are coordinately controlled by inositol and choline.
Results from a deletion analysis of the 5' untranslated region of INOl show there are cis-acting regulatory sites that act to reduce transcription of this gene under repressing growth conditions. See Hirsch, J. P., Lopes, J. M., Chorgo, P. A., and Henry, S. A. (1991). Nucl. Acids Res. Submitted. In order to understand the mechanism by which the OPIl gene, SEQ ID NO: 1, and its product, SEQ ID NO: 2, interact with other regulatory genes and the structural genes under their control, a detailed molecular analysis of the OPIl gene, SEQ ID NO: 1, has been achieved. The present invention presents the genetic mapping, cloning and molecular analysis of the opil gene, SEQ ID NO: 1, and the use of it and its gene product, SEQ ID NO:2, in phospholipid biosynthesis to excrete excess inositol, for instance, for human or animal consumption.